We propose to map major core histone variants throughout the annotated Drosophila melanogaster and Caenorhabditis elegans genomes at high resolution. These maps will provide a chromatin-based framework for interpreting epigenetic information and gene expression patterns determined by others. To accomplish this, we will use the biotin-tagging system that we have successfully applied for genome-scale chromatin affinity purification together with a standard microarray readout platform. In both D. melanogaster and C. elegans we will profile the universal replication-independent histone 3 variant, H3.3, as well as the control replication-coupled variant, H3. These data will provide high-resolution genome-wide maps of histone dynamics in both model organisms. The close correspondences of H3.3 patterns to patterns of active histone modifications genome-wide and with DNasel hypersensitive sites at homeotic gene clusters suggests that our genome-wide maps can be used to validate efforts aimed at mapping these other genomic features. In both D. melanogaster and C. elegans we will also profile the H2AZ variant, which has been linked to epigenetic processes in a variety of different organisms. Drosophila H2AZ plays a unique dual role as the H2AX variant, called H2AV, and our preliminary studies indicate that patterns of H2AV differ from those of H3.3, thus providing a different epigenetic profile of histone dynamics genome-wide. To expand our understanding of the functional roles of both classes of histone variants, we will use RNAi to knock down the function of key chromatin regulators in Drosophila cell lines and in whole C. elegans at different developmental stages. Transcriptional profiling after knock-down of the variants and their assembly machines will provide functional validation of roles that they play in gene regulation, and histone variant profiling after knock-down should help to identify target genes. Together with other types of genome-wide information our histone variant maps are likely to contribute to a fuller understanding of epigenetic regulatory elements in the genomes of these two model organisms. Given that the same histone variants are thought to play similar roles in essentially all complex eukaryotes, our basic approach can be immediately applied to the human genome.